Abstract
Translating Ribosome Affinity Purification (TRAP) is a technology to isolate the population of mRNAs associated with at least one 80S ribosome, referred as the translatome. TRAP is based on the expression of an epitope-tagged version of a ribosomal protein and the affinity purification of ribosomes and associated mRNAs using antibodies conjugated to agarose beads. Quantitative assessment of the translatome is achieved by direct RNA sequencing (RNA-SEQ), which provides accurate quantitation of ribosome-associated mRNAs and reveals alternatively spliced isoforms. Here we present a detailed procedure for TRAP, as well as a guide for preparation of RNA-SEQ libraries (TRAP-SEQ) and a primary data analysis. This methodology enables the study of translational dynamic by assessing rapid changes in translatomes, at organ or cell-type level, during development or in response to endogenous or exogenous stimuli.
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Acknowledgements
We thank Ankelika Mustroph, Cristina Branco-Price, and others that have contributed to developing the TRAP-SEQ technology. Sequencing of the M. truncatula TRAP libraries was done at John Craig Venter Institute. We also thank Christopher Town and Benjamin Rose for discussion and advice on RNA-SEQ analysis. This work has been financially supported by PICT 2007-00095 and PICT 2010-2431, ANPCyT, Argentina, funded to M.E.Z. and by an International cooperation program of CONICET, Argentina, and the NSF, USA, funded to M.E.Z. and J.B.S.
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Reynoso, M.A., Juntawong, P., Lancia, M., Blanco, F.A., Bailey-Serres, J., Zanetti, M.E. (2015). Translating Ribosome Affinity Purification (TRAP) Followed by RNA Sequencing Technology (TRAP-SEQ) for Quantitative Assessment of Plant Translatomes. In: Alonso, J., Stepanova, A. (eds) Plant Functional Genomics. Methods in Molecular Biology, vol 1284. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2444-8_9
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DOI: https://doi.org/10.1007/978-1-4939-2444-8_9
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